Estrogen regulation of methyl p-hydroxyphenyllactate hydrolysis: correlation with estrogen stimulation of rat uterine growth

We have recently demonstrated that methyl p-hydroxyphenyllactate (MeHPLA) is the endogenous ligand for nuclear type II binding sites in the rat uterus and other estrogen target and non-target tissues. MeHPLA binds to nuclear type II binding sites with a very high binding affinity (Kd approximately 4-5 nM), blocks uterine growth in vivo, and inhibits MCF-7 human breast cancer cell growth in vitro. Conversely, the free acid (p-hydroxyphenyllactic acid, HPLA) interacts with type II binding sites with a much lower affinity (Kd approximately 200 nM) and does not inhibit estrogen-induced uterine growth in vivo or MCF-7 cell growth in vitro. On the basis of these observations, we suggested that one way that estrogen may override MeHPLA inhibition of rat uterine growth may be to stimulate esterase hydrolysis of MeHPLA to HPLA. The present studies demonstrate that the rat uterus does contain an esterase (mol. wt approximately 50,000) which cleaves MeHPLA to HPLA, and that this enzyme is under estrogen regulation. This conclusion is supported by the observations that MeHPLA esterase activity is increased 2-3-fold above controls within 2-4 h following a single injection of estradiol, and is maintained at high levels for 16-24 h following hormone administration. This sustained elevation of MeHPLA esterase activity correlates with estradiol stimulation of true uterine growth and DNA synthesis.     

Cytosol type II sites in the rat uterus: interaction with an endogenous ligand

Previous studies from our laboratory demonstrated that normal, but not malignant tissues, contain a ligand which competes for [3H]estradiol binding to nuclear type II sites in the rat uterus. Since elevated nuclear levels of type II sites are correlated with estrogen stimulation of uterine growth and DNA synthesis, we believe this ligand may regulate cell growth. The present studies show that the ligand for nuclear type II sites also interacts with type II sites in uterine cytosol. This was demonstrated by dilution experiments which show that greater quantities of type II sites are measured in dilute (10 mg/ml) than in concentrated (40 mg/ml) uterine cytosol. Furthermore, stripping of uterine cytosol with 1% dextrancoated charcoal, or pre-binding cytosol type II sites to hydroxylapetite (HAP) prior to binding analysis, removed the ligand from these preparations such that high levels of type II sites were measured. Following charcoal stripping, cytosol type II sites demonstrated good specificity for estrogenic hormones but not progesterone, corticosterone, or the triphenylethylene anti-estrogen, nafoxidine. Since the level of type II sites in the cytosol always preceded and exceeded the level of this site measured in uterine nuclei at all times following estrogen treatment (0-96 h), we believe cytosol type II sites may function as an type II-ligand binding protein (LBP) which regulates the availability of the ligand for interaction with nuclear type II sites. This is consistent with our observation that type II sites are not depleted from uterine cytosol by estrogen treatment and nuclear type II sites are very tightly associated with the nuclear matrix.     

Bioflavonoid interaction with rat uterine type II binding sites and cell growth inhibition

Competition analysis with a number of known bioflavonoids demonstrated that these compounds (luteolin, quercetin, pelargonin) compete for [3H]estradiol binding to cytosol and nuclear type II sites in rat uterine preparations. The inhibition of [3H]estradiol binding to type II sites was specific and these bioflavonoids did not interact with the rat uterine estrogen receptor. Since estradiol stimulation of nuclear type II sites in the rat uterus is highly correlated with cellular hypertrophy and hyperplasia, we assessed the effects of these compounds on the growth of MCF-7 human breast cancer cells in culture and on estradiol stimulation of uterine growth in the immature rat. The data demonstrated that addition of quercetin (5-10 micrograms/ml) to MCF-7 cell cultures resulted in a dose-dependent inhibition of cell growth (DNA/flask). This effect was reversible by removal of quercetin from the culture medium, or by the addition of 10 nM estradiol-17 beta to these cell cultures containing this bioflavonoid. Since estradiol-17 beta (10 nM) stimulated nuclear type II sites and proliferation of MCF-7 cells, we believe bioflavonoid inhibition of MCF-7 cell growth may be mediated through an interaction with nuclear type II sites. This hypothesis was confirmed by in vivo studies which demonstrated that injection of luteolin or quercetin blocked estradiol stimulation of nuclear type II sites in the immature rat uterus and this correlated with an inhibition of uterine growth (wet and dry weight). These studies suggest bioflavonoids, through an interaction with type II sites, may be involved in cell growth regulation.     

Uterine type II estrogen-binding sites are not of eosinophil origin

A recent report by Lyttle et al. (Lyttle, C. R., Medlock, R. L., and Sheehan, D. M. (1984) J. Biol. Chem. 259, 2697-2700) suggested that nuclear type II sites in the rat uterus are of eosinophil origin and may represent [3H]estradiol binding to eosinophil peroxidase. To further evaluate this hypothesis we examined the response of nuclear type II sites to estrogen under conditions where eosinophils are not present. Results of our experiments show that physiological levels of estradiol-17 beta (10 nM for 72 h) will stimulate nuclear type II sites in highly purified cultures (21-25 days; 4 passages) of rat uterine stromal and myometrial cells. The magnitude of the response of type II sites to estradiol in these stromal (4-fold) and myometrial (80-fold) cell cultures was essentially identical to that observed in the uterine cell types following in vivo estrogen treatment. Since these highly purified cultures of uterine cells were prepared from the uterus of a 21-day ovariectomized rat which is devoid of eosinophils, we conclude that estradiol stimulation of nuclear type II sites is a direct intracellular response to estrogen which occurs independent of eosinophil accumulation. Furthermore, we have found that type II sites in the rat uterus are not peroxidase. This was demonstrated by experiments which show type II sites are present in the 39,000 X g supernatant fraction of uterine cytosol, whereas peroxidase activity is quantitatively recovered in the crude mitochondrial (39,000 X g) pellet. Likewise, the small amount of peroxidase activity (approximately 10%) in the total homogenate which contaminates our nuclear pellet preparations was extracted (98-100%) with 0.5 M CaCl2. Type II estrogen-binding sites (95-100%) remained associated with the nuclear pellet fraction after peroxidase extraction. Therefore, stimulation of cytosol and nuclear type II sites by estrogen in the rat uterus is a direct intracellular response to the hormone unrelated to eosinophil accumulation and/or peroxidase activity.     

Estrogen receptors in the rat uterus. Retention of hormone-receptor complexes.

The retention pattern and biochemical characteristics of estrogen receptors in the nuclei of uterine cells were studied as a function of time after the in vivo injection of estradiol (E2) to immature female rats. One hour after the injection of 0.1 mug of tritiated E2, approximately 0.20 pmol per uterus of receptor bound hormone is retained in uterine nuclei. This dose of E2 produces a maximal uterotrophic response. Six hours after E2 administration, uterine nuclei retain 0.04-0.08 pmol of hormone per uterus. Hormone receptor complexes extracted from uterine nuclei 1, 3, and 6 h after in vivo injection of hormone have similar structural and binding characteristics. Receptors extracted at all three times sediment at 5S in high salt gradients and have a dissociation binding constant of approximately 3 nM for E2. The wash-out curves of receptors as a function of salt concentration are identical for uterine nuclei from animals treated for 1 or 6 h with estradiol, suggesting that the nature of the nuclear binding of receptors is not altered during this time interval. Experiments utilizing the injection of unlabeled estradiol, followed by an in vitro exchange procedure with tritiated estradiol, indicated that the total nuclear estrogen receptor sites, i.e., filled and vacant, decreased similarly.     

Estrogen dependent induction of low affinity binding sites in the nuclear fraction of rat uterus

Type II estradiol binding sites characterized by lower affinity and higher capacity than type I receptor sites have been described in rat uterine nuclei. These sites appeared to be dependent on estrogen stimulation. Reducing agents prevented estradiol binding to these sites. In the present study, the situation prevailing in adult rats (Ad) was studied and compared to ovariectomized (Ox) and ovariectomized estrogen prestimulated rats (OxPS). Nuclear precipitate from Ad, Ox and OxPS rats were incubated with tritiated estradiol (E2(3)H) in the presence and in the absence of mercaptoethanol as reducing agent. In the presence of mercaptoethanol, saturation was attained at E2(3)H concentrations above 16 nM. In the absence of reducing agents, a secondary binding was observed in Ad and OxPS which was not saturated at E2(3)H levels up to 80 nM. Non-specific binding obtained with paired aliquots containing 100-fold excess of DES as competitor was not linear but showed a saturation profile, distorting the saturation curve of the specific sites, obtained by subtracting non-specific from total E2(3)H binding. Increasing DES concentrations up to 10,000 nM did not allow to reach complete exchange with E3(3)H ligand bound to specific sites, preventing measurement of binding sites concentration. Incubation of nuclear fractions with increasing concentrations of E2(3)H (up to 6,000 nM) gave a saturation curve with a linear kinetics above 1-2,000 nM, which represented saturation concentration of the specific sites. From this, non-specific and specific moieties could be estimated. Binding capacity of specific sites was of the order of 50-80 pmol uterus. Half saturation was attained between 300 and 600 nM E2(3)H, which approximated the Kdiss of these sites, at variance with the Kdiss of 15-30 nM originally reported for type II binding sites. In conclusion, these results show that secondary binding sites were present in uterine nuclei of Ad and OxPS rats. Binding capacity was about 30-fold higher than that of type I sites. Affinity was however very low, and casts some doubt on the role of these sites as active estradiol binders in physiological situations. Their increase under the influence of estrogen may however be related to some as yet undetermined role.     

Heterogeneity of Estrogen Binding Sites in Mouse Mammary Cancer

Abstract

The recent demonstration in our laboratory of at least two specific estrogen binding sites in the rat uterus prompted us to investigate similar heterogeneity of binding sites in a trans-plantable ovarian dependent mouse mammary tumor (MXT-3590). Saturation analysis of cytoplasmic (protamine sulfate or hydroxylapatite exchange assay) or crude nuclear fractions (protamine sulfate precipitated nuclear exchange assay) revealed two binding components: type I which conforms to the classically described estrogen receptor and type II which has a lower affinity for estradiol but a greater capacity than type I sites. Exposure of cytosol to charcoal partially removes bound ³H-estradiol from type II sites but not from type I sites. Type II sites are specific for estrogens and do not translocate from the cytoplasmic to the nuclear compartment. Although Type II sites undergo dissociation on prelabeled sucrose density gradients, they are readily demonstrable by postlabeling sucrose density gradient fractions and hydroxylapatite adsorption. Since the presence of type II sites interferes with the measurement of the estrogen receptor (type I) which may also undergo dissociation on sucrose gradients, we recommended that the technique of postlabeling be used for the sucrose gradient analysis of type I and II sites. In addition, saturation assays should be performed over a wide range of ³H-es-tradiol concentrations (0.1–120 nM) for proper evaluation of both sites. These considerations may contribute to more accurate predictions about the response of breast cancers to endocrine therapies.     

Preliminary characterization of an endogenous inhibitor of [3H]estradiol binding in rat uterine nuclei

The rat uterus contains two classes of specific nuclear estrogen-binding sites which may be involved in estrogen action. Type I sites represent the classical estrogen receptor (Kd = 1 nM) and type II sites (Kd = 10-20 nM) are stimulated in the nucleus by estrogen under conditions which cause uterine hyperplasia. Dilution of uterine nuclear fractions from estrogen treated rats prior to quantitation of estrogen binding sites by [3H]estradiol exchange results in an increase (3- to 4-fold) in the measurable quantities of the type II site. Estimates of type I sites are not affected by dilution. These increases in type II sites following nuclear dilution occur independently of protein concentration and result from the dilution of a specific endogeneous inhibitor of [3H]estradiol binding to these sites. The inhibitor activity is present in cytosol preparations from rat uterus, spleen, diaphragm, skeletal muscle, and serum. Preliminary characterization of the inhibitor activity by Sephadex G-25 chromatography shows two distinct peaks which are similar in molecular weight (300). These components (alpha and beta) can be separated on LH-20 chromatography since the beta-peak component is preferentially retained on this lipophilic resin. Partial purification of the LH-20 beta inhibitor component by high performance liquid chromatography and gas-liquid chromatography-mass spectrometric analysis suggests the putative inhibitor activity is not steroidal in nature and consists of two very similar phenanthrene-like molecules (molecular weights 302 and 304). Analysis of cytosol preparations on LH-20 chromatography shows that non-neoplastic tissues (uterus, liver, lactating mammary gland) contain both and inhibitor components whereas estrogen-induced rat mammary tumors contain very low to nonmeasurable quantities of the beta-peak inhibitor activity.     

Concepts related to salt resistant estradiol receptors in rat uterine nuclei: nuclear matrix

When ³H-estradiol (0.1 μg) is injected into immature female rats, virtually all of the label that is recovered with uterine nuclei can be solubilized by 0.6 M KCl. Salt resistant uterine nuclear estrogen binding sites do not become labeled within one hour after the injection of ³H-estradiol, but these sites do exist and can be revealed when isolated nuclei are subjected to an $\text{in vitro}$ estradiol exchange assay. These saturable, high affinity salt resistant sites appear to be associated with the uterine nuclear matrix, a residual structure of the nucleus.     

Estradiol-17β receptors in the immature rat ovary

Estradiol binding components in the cytosol and nuclear fractions of the ovary from immature rats (22–28 days old) were characterized by $\text{in vitro}$ methods. Several of the biochemical characteristics of the estradiol binding components in the ovarian tissue were compared with the estradiol receptor from the uterus. The results suggest that the ovarian estradiol binding components are similar to the specific high affinity estradiol receptors in the uterus. In the cytosol of intact rat ovary a significant fraction of the total binding sites was found to be occupied, presumably by the endogenous estrogen. Following hypophysectomy there was a significant increase in the available cytosol binding sites. Evidence for translocation of cytosol receptor-estrogen (RE) complex to the nucleus was obtained for the ovary. The sedimentation properties of the RE complex of the ovary and the uterus are similar. The ovarian cytosol RE complex sediments at 7-8S in glycerol gradients at low ionic strength and at 4S in sucrose gradients at high ionic strength. Following extraction with 0.4 M KCl the ovarain nuclear RE complex sediments at 5S in sucrose gradients which is identical to that of the uterine nuclear receptor.     

Cytosolic Type II Estrogen Binding Site in Rat Uterus: Specific Photolabeling with Estrone

Abstract

A low affinity (Kd = 30 nM), large capacity (Bmax = 2.6 pmol/g tissue) estrogen binding site was photolabeled from estradiol-stimulated rat uterus cytosol. To maximize levels of this binding site and reduce those of the type I binding site, ovariectomized rats were injected with high doses of estradiol (10μg per day) for four days with the last injection two hours before sacrifice. This treatment depleted type I estrogen receptors from the cytosol (by 90%) and raised levels of type II sites in the nucleus without affecting cytosolic type II levels. The type II estradiol binding sites were distinguished from the type I sites on the basis of their dissociation kinetics, pH-sensitivity and their behavior towards potassium chloride, somatostatin, sodium thiocyanate, sulfhydryl reagents and ammonium sulfate precipitation. These type II binding sites could be covalently photolabeled with tritiated estrone. A molecular weight of 43 kDa was found on SDS PAGE.     

Heterogeneity of [3H]estradiol binding sites in the rat prostate: properties and distribution of type I and type II sites

In order to assess the rat prostate as a target tissue for receptor-mediated estrogen action, we have studied the properties and distributions of estrogen binding sites in the dorsolateral (DLP) and ventral (VP) prostate. Saturation analyses over a wide range of [3H]estradiol ([3H]E2) concentrations (0.5-100 nM) revealed two distinct types of binding sites in the cytosol and nuclear fractions of DLP of intact rats. The high affinity (type I) estrogen binding sites saturated at 2-4 nM of [3H]E2 and had a capacity of 170 fmol/mg DNA in the cytosol and 400 fmol/mg DNA in the nuclei. DLP type I sites had ligand specificity similar to that described for the classical estrogen receptors (ERs) found in female target tissues. The moderate affinity (type II) estrogen binding sites saturated at 15-30 nM of [3H]E2 and had a capacity of 850 fmol/mg DNA in the cytosol and 1600 fmol/mg DNA in the nuclei. DLP type II sites shared some characteristics of the type II ERs described for the rat uterus; they were estrogen specific, heat labile, and sensitive to reducing agents such as dithiothreitol. Saturation analyses on VP cytosols and nuclear fractions revealed only high affinity sites but no moderate affinity sites in the tissue preparations. Our finding that prostatic type II estrogen binding sites are present exclusively in the DLP supports the concept that basic biological differences exist between the two major prostatic lobes of the rat. Furthermore, our findings may help elucidate the observed differences in susceptibility between these two lobes to the hormonal induction of proliferative prostatic lesions.     

Radioautographic study of the effect of estradiol-17 , estrone, estriol, progesterone, testosterone and corticosterone on the in vitro uptake of 2,4,6,7- 3 H estradiol-17 by uterine eosinophils of the rat

The in vitro uptake of 2,4,6,7-tritiated estradiol-17beta in uterine eosinophils of the rat was inhibited by the presence of nonradioactive estradiol-17beta, estrone, and estriol, but not by progesterone, testosterone, or corticosterone. This action is attributed to competition between tritiated estradiol and the various estrogenic compounds for the same binding site. Compounds without any estrogenic activity do not compete. The proposal is made that the eosinophil binding system and the 8S-5S binding system are involved in different mechanisms of estrogen action. The parallelism between the doses of estradiol and estriol needed to promote certain estrogenic early effects in the uterus, and the affinity of these steroids for the eosinophil uptake sites, suggests that uterine eosinophils might be responsible for some of these early effects, such as water imbibition, histamine releasing activity, and estrogen priming effect.     

Properties of estradiol binding components in hamster uterine nuclei

Only one estrogen-binding component (Type I) was observed in salt (0.5 M KCl) extracts of proestrous hamster uterine nuclei. In addition to the classical estrogen receptor (Type I), a second binding component (Type II) was detected by [³H]estradiol exchange assay performed with hamster uterine nuclear suspensions. Although this Type II binder was not detected in salt extracts, a similar binding component was found in the nuclear debris remaining after salt extraction. The Type II binding component in the nuclear debris did not posess estrogen-binding specificity. Lack of specificity for estrogens, resistance to KCl extraction, and high capacity differentiated this Type II binder from the classical estrogen receptor. Preparation of nuclear fractions in buffer containing glycerol and monothioglycerol resulted in greater recovery of nuclear estrogen receptor (Type I) as compared to buffer lacking these constituents.     

Nuclear estradiol receptors in several "non-target" organs of rats]

Some properties of the macromolecules of the KCl-extracts of the nuclei of the uterus, kidney, liver, testis and prostate, specifically binding estradiol (E2), were studied. These macromolecules of the uterus and the liver were found to be maximally extracted from chromatin by the 0.6 M KCl concentration. The capacity of the macromolecules of the uterine, kidney and liver nuclear extracts to bind E2 specifically is destroyed completely by pronase, but not by RNA-ASe and DNA-ase, pointing to the protein nature of these macromolecules. Only estrogenic compounds, but not testosterone, 5alpha-dihydrotestosterone, progesterone or corticosterone were capable to compete with H3--E2 for the E2--binding sites of the macromolecules of the nuclear extracts of all the organs investigaeted. It is assumed that macromolecules of the nuclei of the investigated nontarget organs specifically binding E2 are estrogen receptors.     

Concentration and turnover of estradiol in the rat uterus in vivo

The concentrations and turnover of estradiol isolated from cytosolic and nuclear fractions of uteri from ovariectomized rats given estradiol, either in single injections or in continuous infusion, were analyzed by gas chromatography-mass spectrometry. The analytical method was validated for different organs and lower limits of analysis were established. After infusion of 20 ng x h-1 for 18-22 h, mean estradiol levels were 2.0-2.4 fmol x mg-1 uterine wet weight in the nuclear fraction, and 1.2-1.5 fmol x mg-1 in the cytosolic fraction. The concentrations were about five times higher after a single injection of one microgram estradiol but the distribution between nuclear and cytosolic fractions was almost the same. The concentrations of estradiol in nuclei from liver and spleen were 50-200 times lower than those in uterus. Taken together with previous knowledge, the results indicate that the distributions of estradiol and its receptor are not the same and that hormone response cannot be predicted from the concentration of receptors alone. The exchange of estradiol molecules in the uterus was followed after a change of the infusion from unlabelled to [11,12,12-2H3]-labelled estradiol, or vice versa. The uterine uptake of estradiol was calculated to be about 0.7 fmol x h-1 x mg-1 uterine wet weight. The half-life time was calculated to be at least 4 h for estradiol molecules isolated from the nuclear fraction and 3 h (significantly shorter) for those isolated from the cytosolic fraction. The results indicate an uptake of 40-90% of all estradiol passing through the uterus in proestrus with only about 10% of available receptors becoming occupied. When the infusion was changed from estradiol to ethynylestradiol, estradiol disappeared from the uterus at the same rate as in the experiments above. Ethynylestradiol was taken up at a rate of about 0.3-0.4 fmol x h-1 x mg-1 tissue. The percentage of total steroid found in the nuclear fraction was higher for ethynylestradiol, about 70%, than for estradiol, about 60%, indicative of a more stable association of receptor to nuclear binding sites when ethynylestradiol is the ligand.     

Interaction of the calf uterine estrogen receptor with acceptor sites in heterologous chicken target cell nuclei

Estrogen receptor (ER) from chicken liver and calf uterus were used to study the capacity and the characteristics of the receptor binding sites (acceptor sites) in chicken target cell nuclei. Binding studies were performed at a physiological salt concentration of 0.15 M KCl. Binding of liver ER to liver nuclei was temperature-dependent, showing a 9-fold increase between 0 and 28 degrees C. The maximal number of acceptor sites measured in this cell-free system (280 sites/nucleus) was considerably lower than measured in nuclei after in vivo administration of estrogen (820 sites/nucleus). Moreover incubation of nuclei with the liver ER preparation resulted in a substantial breakdown of nuclear DNA, making this ER less suitable for DNA binding studies. The temperature-activated calf uterine receptor bound to liver nuclei at 0 degrees C, at which temperature no DNA degradation was measured. To all chicken cell nuclei tested, the receptor bound with a high affinity (Kd = 0.4-1.0 nM). Nuclear binding displayed tissue specificity: oviduct greater than heart, liver greater than spleen greater than erythrocytes and was salt dependent. Calf uterine ER binding in liver nuclei ranged from 3000-6000 acceptor sites per nucleus when assayed under conditions of a constant protein or a constant DNA concentration. Nuclei isolated from estrogen-treated cockerels bound a 2-fold lower number of calf uterine ER complexes when compared to control nuclei. Incubation of nuclei with a fixed concentration of [3H]ER from liver and increasing concentrations of uterine non-radioactive-ER also resulted in a reduced binding of the liver receptor. Both types of experiments suggest that liver and uterine ER compete for a common nuclear acceptor site. Our data demonstrate that the ER from calf uterus is very useful as a probe to examine the nature of the acceptor sites in heterologous chicken target cell nuclei. The assay system functions at 0 degrees C, a temperature at which no DNA degradation occurs.     

Dietary phytoestrogens and cancer: in vitro and in vivo studies

Thirty postmenopausal women (11 omnivores, 10 vegetarians and 9 apparently healthy women with surgically removed breast cancer) were investigated with regard to the association of their urinary excretion of estrogens, lignans and isoflavonoids (all diphenols) with plasma sex hormone binding globulin (SHBG). A statistically significant positive correlation between urinary total diphenol excretion and plasma SHBG was found which remained statistically significant after elimination of the confounding effect of body mass determined by body mass index (BMI). Furthermore we found a statistically significant negative correlation between plasma SHBG and urinary excretion of 16α-hydroxyestrone and estriol which also remained significant after eliminating the effect of BMI. Furthermore we observed that enterolactone (Enl) stimulates the synthesis of SHBG by HepG2 liver cancer cells in culture acting synergistically with estradiol and at physiological concentrations. Enl was rapidly conjugated by the liver cells, mainly to its monosulfate. Several lignans and the isoflavonoids daidzein and equol were found to compete with estradiol for binding to the rat uterine type II estrogen binding site (the s.c. bioflavonoid receptor). It is suggested that lignans and isoflavonoids may affect uptake and metabolism of sex hormones by participating in the regulation of plasma SHBG levels and in this way influence their biological activity and that they may inhibit cancer cell growth like some flavonoids by competing with estradiol for the type II estrogen binding sites.     

Multiple species of estrogen binding sites in the nuclear fraction of the rat prostate

Specific estrogen-binding sites have been demonstrated in purified nuclear fractions of prostates from intact rats. Saturation analysis of nuclei over a wide range of [³H]-estradiol concentrations (0.15 to 90 nM) has shown two different types of binding sites: a) one with high affinity (Kd of 0.5–0.8 nM) and low capacity for estradiol (approximately 162 fmole/mg DNA); b) a second with a lower affinity (Kd of 30–40 nM), which shows a higher capacity (approximately 860 fmole/mg DNA), and displays a saturation curve that is sigmoidal and that appears to be similar to those for Type II estrogen-binding sites in rat uterus. These results suggest that the actions of estradiol in the prostate are mediated by specific nuclear binding sites.